Fig. 2. Topology of Vtc proteins. (A) Protease digestion of vacuoles carrying Vtc1p-GFP or Vtc3p-GFP*. Vacuoles were isolated from BJ3505 cells expressing Vtc1p-GFP (from plasmid pYER-GFP) or from SBY593 cells expressing chromosomally encoded Vtc3p-GFP*. 20 µg vacuoles (0.1 mg/ml in PS buffer) were incubated with 10 µg/ml proteinase K in the presence or absence of 0.5% (w/v) Triton X-100 (5 minutes, 0°C). Digestion was stopped by adding one volume of 2 mM PMSF in PS buffer. Proteins were TCA precipitated, washed with acetone and solubilized in 100 µl non-reducing SDS-sample buffer. Coprecipitated Triton X-100 that can interfere with SDS-PAGE was extracted with chloroform/methanol (water:chloroform:methanol 2:1:2). Pellets were resolubilized in 50 µl reducing SDS-sample buffer, split and analyzed by 15% and 7.5% gels and western blotting with rabbit anti-GFP, goat anti-Vtc4p or mouse anti-Pho8p. TM, transmembrane fragment; pPho8p, pro-Pho8p; mPho8p, mature Pho8p; pPho8p-cytD, pPho8p fragment lacking the cytosolic tail. (B) Topology of the Vtc proteins and of Pho8p.